1019 Erlotinib Potentiates Cetuximab-dependent Cell Citotoxicity in Egfr Wild Type Nsclc Cell Lines

Cavazzoni et al. Molecular Cancer 2012, 11:91http://www.molecular-cancer.com/content/11/1/91

RESEARCH Open Access

Combined use of anti-ErbB monoclonal antibodiesand erlotinib enhances antibody-dependentcellular cytotoxicity of wild-type erlotinib-sensitiveNSCLC cell linesAndrea Cavazzoni1†, Roberta R Alfieri1*†, Daniele Cretella1, Francesca Saccani1, Luca Ampollini2, Maricla Galetti1,3,Federico Quaini1, Gallia Graiani1, Denise Madeddu1, Paola Mozzoni1,3, Elena Galvani1, Silvia La Monica1,Mara Bonelli1, Claudia Fumarola1, Antonio Mutti1, Paolo Carbognani2, Marcello Tiseo4, Elisabetta Barocelli5,Pier Giorgio Petronini1 and Andrea Ardizzoni4

Abstract

Background: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment indifferent tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelialcancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonalantibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proveneffective in the treatment of advanced NSCLC.

Results: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab toimprove the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observedto increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to thedrug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedlyincreased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, thecombination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone.

Conclusion: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKIsensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograftmodels. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve thetreatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Keywords: Lung cancer, EGFR, Erlotinib, Cetuximab, ADCC

BackgroundThe epidermal growth factor receptor (EGFR, ErbB1,HER1) is the prototypic member of the ErbB family ofreceptor tyrosine kinases (TKs), which further consistsof ErbB2-4 (HER2-4). The ErbB receptors share a similarprotein structure, consisting of an extracellular ligandbinding domain, a single transmembrane domain and an

* Correspondence: [email protected]†Equal contributors1Department of Clinical and Experimental Medicine, University of Parma,Parma, ItalyFull list of author information is available at the end of the article

© 2012 Cavazzoni et al.; licensee BioMed CentCommons Attribution License (http://creativecreproduction in any medium, provided the or

intracellular C-terminal domain with tyrosine kinaseactivity [1]. Upon specific binding of EGF-like ligands tothe extracellular domain, ErbB receptors dimerize, eitheras homo- or heterodimers, and undergo autophosphory-lation at specific tyrosine residues within the intracellu-lar domain. The phosphorylated tyrosines serve asdocking sites for adapter molecules, such as Grb2 andthe p85 subunit of PI3K, which activate a complexdownstream network. The activated signaling pathways,including the Ras/MAPK, Akt/mTOR kinase and STATcascades, in turn regulate transcription factors and otherproteins involved in cell proliferation, survival, motility

ral Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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and differentiation [2]. Two main strategies targetingErbB receptors have been developed: small-moleculeinhibitors of the tyrosine kinase domain (EGFR tyrosinekinase inhibitors [TKIs], such as erlotinib and gefitinib),and monoclonal antibodies (such as cetuximab, anti-EGFR and trastuzumab, anti-HER2), directed against theextracellular domain, which inhibit phosphorylation/activation and promote internalization. EGFR and HER2are overexpressed in 40-80% and 25-30%, respectively, ofnon-small cell lung cancer (NSCLC) patients and theiroverexpression has been frequently correlated with apoor prognosis [3,4].Erlotinib is an effective treatment for NSCLC patients

and has been registered as a second and third-line treat-ment of NSCLC regardless of EGFR mutation status [5].Gefitinib has been registered for the therapy of

advanced NSCLC harbouring activating EGFR mutationsin the tyrosine kinase domain, the most frequent beingL858R in exon 21 and Del (746–750) in exon 19 [6].Although mutations in EGFR are useful predictors forthe activity of EGFR-TKI, they cannot be used as theonly criterion to determine who should receive anti-EGFR therapy and it is becoming increasingly clear thateven patients with EGFR wild-type can benefit fromEGFR-TKI [5,7,8].Cetuximab is a chimeric IgG1 monoclonal antibody

(mAb) that blocks ligand binding to EGFR, leading to adecrease in receptor dimerization, autophosphorylation,and activation of signaling pathways [9]. In addition thebinding of cetuximab initiates EGFR internalizationand degradation which leads to signal termination.Moreover, unlike EGFR-TKIs, cetuximab can induceantibody dependent cellular cytotoxicity (ADCC)activity, an important immunologic antitumour effect.Cetuximab in combination with chemotherapy hasbeen approved by the FDA for the treatment of meta-static colorectal cancer and of locally advanced headand neck cancer.Two randomized phase III trials in NSCLC patients,

evaluating cetuximab in addition to first-line chemo-therapy, showed a small benefit in overall survival forthe experimental treatment, which was considered in-sufficient by the EMA for marketing approval [10,11].However, a subgroup analysis of the FLEX phase IIItrial recently demonstrated a larger survival benefitfrom the experimental treatment in patients with highimmunohistochemical EGFR expression [12].Trastuzumab, registered for the treatment of HER2

positive breast cancer, has also been tested in phase IItrials as a single agent and in combination with cytotoxicchemotherapy for patients with NSCLC. These trialshave not yet produced any convincing evidence of animproved antitumour activity by adding trastuzumab tostandard chemotherapy in NSCLC [13,14].

Several preclinical studies on cell lines from differenttumour types, indicated that the association betweenEGFR/HER2 mAbs with TKIs displays an increased effi-cacy [15].In this study we explored the potential of combining

erlotinib with either cetuximab or trastuzumab in orderto improve the efficacy of EGFR targeted therapy inEGFR wild-type sensitive NSCLC cell lines. Our resultsindicate that EGFR-TKI increases surface expression ofEGFR and/or HER2 only in erlotinib sensitive NSCLCcell lines and, in turns, leads to increased susceptibilityto ADCC both in vitro and in xenograft models.

ResultsDifferential effects of erlotinib on EGFR and HER2expression in sensitive and resistant NSCLC cell linesFirstly, we evaluated the effect of erlotinib on total EGFRand HER2 protein levels in sensitive NSCLC cell lines(Calu-3, H322 and H292 cell lines carrying wild-typeEGFR; PC9 and HCC827 carrying EGFR E746-A750delmutation) and in resistant cell lines (A549, H1299,H1703 and Calu-1 intrinsically resistant carrying wild-type EGFR; HCC827GR5 with MET amplification asmechanism of acquired resistance to TKI) [16]. Asshown in Figure 1A, erlotinib induced accumulation ofEGFR protein in Calu-3 and H322 cells while HER2accumulated in H322, H292, PC9 and HCC827 cells in adose-dependent manner. The EGFR/Actin and HER2/Actin ratios obtained after treatment at 1 μM or 10 nMerlotinib were calculated and values expressed as folddifferences versus control (Figure 1B). In contrast, EGFRand HER2 protein accumulation was not observed inany cancer cell line with intrinsic resistance to EGFRinhibitors until the concentration of 10 μM. Indeed theratios EGFR/Actin or HER2/Actin were similar or evenlower than those calculated in untreated cells (Figure 1C)and similar results were obtained with gefitinib (notshown). A representative Western blotting of resistantH1299 cell line is reported in Figure 1D.The different effect of TKIs on HER2 expression be-

tween sensitive and resistant NSCLC cell lines was con-firmed in the HCC827 parental and in the HCC827GR5resistant clone treated for 48 h with gefitinib (Figure 1E).

Erlotinib increases the cell surface expression of EGFRand HER2 in erlotinib sensitive NSCLC cell linesEGFR and HER2 expression on the plasma membranewas quantified by flow cytometry in sensitive EGFRwild-type NSCLC cell lines Calu-3, H322 and H292 afterexposure to 1 μM erlotinib for 24 h. The drug enhancedsurface expression, calculated as molecules of equivalentsoluble fluorophore, of EGFR in Calu-3 (Figure 2A) andH322 (Figure 2C, 2D) and of HER2 in H292 (Figure 2B)and H322 (Figure 2C, 2D) cell lines. In H322 cell line,

Figure 1 (See legend on next page.)

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(See figure on previous page.)Figure 1 Erlotinib induces EGFR and HER2 protein accumulation only in sensitive NSCLC cell lines. (A) Calu-3, H322, H292, PC9 andHCC827 cell lines were treated with the indicated concentrations of erlotinib for 48 h. At the end of the drug treatment cell lysates wereimmunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin andHER2/Actin were calculated at 1 μM erlotinib for Calu-3 H322 and H292 or 10 nM for PC9 and HCC827 and values are expressed as fold increaseversus control (B). (C) HCC827GR5, A549, H1299, H1703, Calu-1 cell lines were treated with 1 μM erlotinib for 48 h and at the end of treatmentcell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios ofEGFR/Actin and HER2/Actin were calculated and values are expressed as fold increase versus control. (D) Representative Western blotting ofresistant H1299 cell line exposed to increased concentration of erlotinib. (E) HCC827 parental cell line and HCC827GR5 resistant clone weretreated with the indicated doses of gefitinib and processed as above. The results are from representative experiments. Each experiment, repeatedthree times, yielded similar results.

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the increase in EGFR and HER2 surface expression wasdose and time dependent (Figure 2C, 2D). Western blotanalysis of isolated cell surface membrane proteins (insetFigure 2A) confirmed the increase of EGFR in erlotinibtreated Calu-3 cells.Exploiting the ability of cetuximab and trastuzumab to

bind EGFR and HER2, we used these mAbs as primaryantibodies for flow cytometry analysis. By this approach,as shown in Figure 3, we confirmed that the surfacedensity of cetuximab and trastuzumab-binding sites, re-spectively, on Calu-3 (Figure 3A), H322 (3B) and H292(3C) cells were increased after 1 μM erlotinib treatment.These results suggest that erlotinib enhanced cell surface

Figure 2 EGFR and HER2 increase at the plasma-membrane level. Calu24 h, H322 cell line was treated with increasing concentration of erlotinib (end of the treatment, cell surface expression of EGFR and/or HER2 were evMolecules of Equivalent Fluorophore [MEF] or as fold increase versus untreprotein membrane level in Calu-3 after treatment with 1 μM erlotinib for 2proteins were pulled down with neutrAvidin beads. The results are from reyielded similar results.

expression of EGFR or HER2 on sensitive NSCLC cells,leading to an increase of mAbs binding to cancer cellsurface.

Erlotinib induces EGFR protein stabilizationThe possibility that the higher EGFR level observed inCalu-3 cells exposed to erlotinib was due to proteinstabilization or increased synthesis was then explored.As shown in Figure 4A, EGFR level increased after 2 hof erlotinib treatment and reached a plateau after 24 h.Furthermore, the maximum level was maintained duringtime in the presence of the drug. However, after 48 h oferlotinib removal, EGFR expression was reduced to level

-3 (A) and H292 (B) cell lines were treated with 1 μM erlotinib forC) or with 1 μM erlotinib for the indicated period of time (D). At thealuated by flow cytometry and the quantification is reported asated control cells (D). Inset Figure 2A: Western blot analysis of EGFR4 h. Whole cells were labeled with biotin and membrane boundpresentative experiments. Each experiment, repeated three times,

Figure 3 Erlotinib induces the increase of cetuximab and trastuzumab binding sites. Calu-3, H322 and H292 cell lines were treated witherlotinib for 24 h. Binding sites were assessed by flow cytometry using cetuximab (Calu-3, H322) and trastuzumab (H292) as primary antibodiesfollowed by PE-anti-human IgG exposure. Binding sites quantification is reported as Molecules of Equivalent Fluorophore [MEF]. The results in A,B, C are from representative experiments. Each experiment, repeated three times, yielded similar results.

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comparable to untreated cells (Figure 4B). Calu-3 werealso treated with erlotinib in the presence of specificinhibitors of mRNA (Actynomicin D) and protein(Cycloheximide) synthesis. As shown in Figure 4C, theerlotinib- induced EGFR protein increase was neitherinfluenced by Actynomicin D nor Cycloheximide treat-ment indicating that the higher level of EGFR after erlo-tinib treatment could be ascribed to post-transcriptional

Figure 4 Erlotinib induces EGFR protein accumulation through proteiof time with 0.5 μM erlotinib. At the end of drug treatments cell lysates wetreated with 0.5 μM erlotinib for 24 h then the drug was removed and druwere immunoblotted to detect the EGFR protein levels. (C) Calu-3 cells we0.1 μg/ml actynomicin D and 2 μg/ml cyclohexymide. At the end of the exproteins. The immunoreactive spots were quantified by densitometric analfold increase versus control. (D) Calu-3 cells were exposed to 0.5 μM erlotinby RT-PCR. The mean values of two independent measurements (± SD) areWestern blotting in Calu-3 cells untreated or treated for 24 h with 1 μM geμM NVP-BKM-120 and NVP-BYL-719 and 100 nM RAD001. The results are fryielded similar results.

mechanisms such as protein stabilization. Moreover, weanalyzed EGFR transcript level by real time PCR aftererlotinib treatment (Figure 4D). Erlotinib did not affectEGFR mRNA level when compared to untreated cells.With the aim to clarify why the increased level of

EGFR was induced only in sensitive cells, we then testedthe effect of EGFR inhibitors (gefitinib, erlotinib, cetuxi-mab, lapatinib) and of inhibitors of MAPK and PI3K/

n stabilization. (A) Calu-3 cells were treated for the indicated periodre immunoblotted to detect EGFR protein. (B) Calu-3 cells wereg-free medium was added for further 24 and 48 h. Then, cell lysatesre treated for 24 h with erlotinib 0.5 μM, in the absence/presence ofperiment, cell lysates were immunoblotted to detect the indicatedysis, ratios of EGFR/Actin were calculated and values are expressed asib for the indicated period of time and the EGFR mRNA was detectedshown. (E) EGFR, p-P70S6K, p-P44/42 and P44/42 were detected byfitinib, erlotinib and lapatinib, 10 μg/ml cetuximab, 10 μM U0126, 1om representative experiments. Each experiment, repeated three times,

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AKT/mTOR signaling transduction pathways on EGFRaccumulation in Calu-3 cell line. Gefitinib, erlotinib,lapatinib significantly inhibited the phosphorylation ofp70S6K and p44/42 and induced a significant increase inEGFR protein level (Figure 4E). The MEK inhibitorU0126 strongly enhanced EGFR expression, in contrastno increase in the EGFR level was observed after incuba-tion with the inhibitors of PI3K/AKT/mTOR pathwaytested (NVP-BKM-120 and NVP-BYL-719 PI3K inhibi-tors and RAD001 mTOR inhibitor).

Effects of erlotinib and cetuximab combined treatmenton NSCLC cell growth and antibody-dependentcell-mediated cytotoxicityWe then investigated the effect of targeting EGFR byboth the TKI erlotinib and the mAb cetuximab in a cellviability assay (Figure 5). We treated Calu-3, H322 andH1299 cells with erlotinib, cetuximab (doses rangedfrom 1 to 50 μg/ml) or the combination based on theschedule erlotinib 24 h followed by the combination oferlotinib with cetuximab for 72 h. As expected Calu-3(Figure 5A) and H322 (Figure 5B) cells were responsiveto erlotinib and cetuximab treatment, whereas H1299

Figure 5 Effect of erlotinib and cetuximab combination on cell viabiliresistant cells (C) were exposed to the indicated concentrations of erlotinibthe combination of erlotinib and cetuximab for 72 h. After the treatments,inhibition of cell viability versus control cells and are mean values of three

(Figure 5C) cells were resistant to both the single regi-mens. Comparing the experimental combination pointswith that expected by the Bliss criterion, an additiveeffect was observed only in the Calu-3 cells. In fact, inthe H322 cells we failed to observe any improvementtreating cells with the combined treatment and H1299remained resistant.Moreover, cell death, evaluated by morphological ana-

lysis, caspase-3 activation and cleavage, was negligibleunder any of the tested treatments at all the time pointsanalyzed (not shown) suggesting that the combinederlotinib-cetuximab treatment exerted a cytostatic andnot a cytotoxic effect.Since the engagement of immune component system

is one of the main mechanisms of the activity of specificmAbs directed to ErbB family members in vivo, weexamined whether erlotinib could enhance cetuximab-or trastuzumab-mediated ADCC by NK cells. As shownrespectively in Figure 6 A-B cetuximab-dependent cyto-toxicity in the presence of IL-2 activated NK cells washigher in Calu-3 and H322 cells previously treated witherlotinib compared with cells treated with cetuximabalone. Similarly, trastuzumab-dependent cytotoxicity was

ty in NSCLC cell lines. Calu-3, H322 sensitive cells (A, B) and H1299for 96 h or cetuximab for 72 h and to erlotinib for 24 h followed bycell viability was assessed by MTT assay. Data are expressed as percentseparate experiments (**P < 0.01 ***P < 0.001).

Figure 6 Erlotinib potentiates antibody dependent cell cytotoxicity. The indicated human NSCLC cell lines were treated with 1 μM erlotinibfor 24 h. After the treatment with erlotinib, 10 μg/ml Cetuximab (A, B, E) or Trastuzumab (C, D) were added to cancer cells seeded with100 U/ml IL-2 activated-NK cells at the ratio of 1:25 and 1:50. After 4 h LDH release was determined as described in Methods section. The resultsare from representative experiments. The experiment, repeated three times, yielded similar results (**P < 0.01 ***P < 0.001).

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higher in H322 and H292 cells (Figure 6 C-D) previouslytreated with erlotinib compared with cells treated withtrastuzumab alone.On the contrary, the combination of erlotinib with cetux-

imab did not significantly modify the mAb dependent cyto-toxicity in H1299 resistant cancer cells.

Figure 7 Antitumour activity of erlotinib and cetuximab on Calu-3 xeand implanted s.c. (right flank) on female BALB/c-Nude mice. Tumours wertreatments started when tumours reached an average volume of 300 mm3

i.p. twice weekly), or erlotinib plus cetuximab were administered for the duvolume ± SEM of 6 mice per group. (**p < 0.01, ***p < 0.001 vs control; #p <followed by Bonferroni post-test).

Effect of erlotinib and cetuximab on Calu-3 xenograftsTo extend our results in vivo, we tested the combinationof erlotinib with cetuximab in a Calu-3 xenograft model(Figure 7). When tumours were well established (14 dayspost-injection, average volume of 300 mm3) micewere randomized into four treatment groups receiving

nografts. Calu-3 cells were suspended in matrigel and sterile PBS (1:1)e allowed to establish growth after implantation for 14 days, and the. Vehicle, erlotinib (25 mg/Kg, orally 5 days/week), cetuximab (2 mg/Kgration of the study. Data are expressed as percent change in tumour0.05, ###p < 0.001 vs erlotinib; §p < 0.05 vs cetuximab; two-way ANOVA

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erlotinib alone, cetuximab alone, the combination, orvehicles as described in the Methods section. Drugtreatments were well tolerated, and no signs of tox-icity were detected during the study. The treatmentwith either erlotinib or cetuximab as single agent delayedtumour growth. However, the significance of the treatmentversus the control was observed only with cetuximab assingle agent or in combination. Interestingly, the treat-ment with the combination of erlotinib plus cetuximabsignificantly inhibited tumour growth when compared toboth the single agent treatments.The histologic analysis of tumour samples showed that

the subcutaneous injection of Calu-3 strikingly reproducedwithin four weeks the morphological features of humanadenocarcinoma (Figures 8A, 8B1-4, 8C-1). Neoplastic epi-thelial cells clearly expressed cytokeratin (Figure 8C-2) andwere organized in secretory glands surrounded by cellular-ized collagen as evidenced by Masson’s trichrome staining(Figure 8C-4). Regressive phenomena and changes in sizeof neoplastic glands together with intense stromal reactionwere observed in histologic samples of tumours from

Figure 8 Hystological analysis of tumours. A: Selected examples of H&Einduced by Calu-3 injection in untreated (C) and erlotinib (Erl), cetuximab ((scale bars: 1 mm). Higher magnification of the same samples are shown omorphological details of the control neoplastic epithelium (1, H&E) expressepidermis (arrowhead). The presence of inflammatory interstitial cells in a c(bluish) surrounding neoplastic glands (purple) in a Erl + Cet treated tumouillustrating the quantitative measurements of neoplastic, inflammatory cells**p < 0.01, vs control; #p < 0.05, ##p < 0.01 vs erlotinib; §p < 0.05 vs cetuxima

treated mice. Interestingly, cetuximab clearly resulted indense inflammatory periglandular infiltrates mostly com-posed of lymphocytes (Figure 8C-3). Thus, the real impactof treatment on tumour mass within the nodules wasassessed by the morphometric analysis of tissue compos-ition. By this quantitative approach, in agreement withgross anatomic measurements, we documented that thecombination of erlotinib with cetuximab was the most ef-fective treatment on tumour growth inhibition (Figure 8D).This contention was further supported by the

immunofluorescence analysis of Ki67 labelling on tumourtissues at the end of the experimental protocol (Figure 9).Erlotinib was able to reduce proliferation of neoplasticcytokeratinpos cells only in association with cetuximabwhereas cetuximab had a negative impact on cycling cellsalso as individual agent. The TUNEL assay indicated that,according with in vitro data, apoptosis was not a signifi-cant ongoing cellular event implicated in the effect of dif-ferent treatments.We have calculated that 0.026+/−0.016% neoplastic

cells were undergoing apoptosis in untreated tumours.

stained sections of the entire subcutaneous xenografted tumourCet) or erlotinib + cetuximab (Erl + Cet) treated BALB/c nude micen corresponding panels in B (scale bars: 500 μm). C: representativeing cytokeratin (2,* brown-immunoperoxidase) that also depicts theetuximab treated tumour (3, H&E) and the intense collagen depositionr (4, Masson’s trichrome) are shown (scale bars: 100 μm). D: Bar graphsand stromal compartments composing the tumours. (*p < 0.05,b).

Figure 9 Immunohistochemical analysis of cellular proliferation. Immunofluorescence images of Ki67 (green) nuclear labelling of cytokeratin(CK, red) positive neoplastic cells in sections of xenografted tumours from an untreated (A) and Erl + Cet treated (B) BALB/c nude mouse. C: bargraph illustrating the effect of the different treatments on the percentage of cycling (Ki67pos) neoplastic cells within the tumour. CTRL: untreated,ERL: erlotinib, CET: cetuximab, COMB: erlotinib + cetuximab. * p < 0.01 vs CTRL.

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Similar low numbers were obtained after Erlotinib orCetuximab single treatment whereas Erl + Cet increasedthe amount of TUNEL positive neoplastic cells althoughreaching a rate of 0.12+/−0.03%. However, we cannot ex-clude that apoptotic cell death may have contributed tothe positive effect of tumor shrinkage at earlier timesafter drug administration.Thus, these experimental observations suggest that

targeting EGFR by the combination of small moleculesand antibodies increases the in vitro and in vivo anti-proliferative activity of both individual agents andseems to be a potent therapeutic strategy againstNSCLC.

DiscussionThe potential for dual-agent molecular targeting of theErbB family, has been clearly demonstrated in pre-clinical models and confirmed on the clinical setting forHER2-targeting agents in breast cancer. However, littleis known about this therapeutic strategy for differenttargets in other tumour types.In our current study we demonstrated that the

combination of erlotinib with cetuximab or trastuzumabmay enhance the antitumour activity of EGFR-TKI inNSCLC cell lines harbouring wild-type EGFR and inxenograft models.The efficacy of the association between an EGFR/

HER2 mAbs with TKIs has been documented in

preclinical studies in several cell lines originating fromdifferent tumour types [15]. In EGFR wild-type H292and A549 NSCLC cell lines, the combination of eithergefitinib or erlotinib with cetuximab was reported to en-hance growth inhibition in comparison to single treat-ment, particularly in the H292 gefitinib sensitive cell line[17]. In the A549 cell line, expressing both EGFR andHER2, the combination of gefitinib with trastuzumabsignificantly inhibited cell growth and proliferation [18].In Calu-3 xenograft models, the combined treatment oferlotinib and pertuzumab showed an enhanced antitu-mour activity [19].A correlation between cetuximab efficacy and EGFR

expression has been reported in preclinical studies [20]and recently confirmed in clinical trials. Thus, the phaseIII FLEX study involving patients with advanced NSCLCshowed a strong correlation between high tumour EGFRoverexpression and the efficacy of adding cetuximab toplatinum based first-line chemotherapy [12].The combination of a TKI and a mAb was explored as a

potential strategy to overcome acquired resistance to first-generation EGFR-TKIs. Kim and colleagues demonstratedthat the combination of lapatinib with cetuximab over-came gefitinib resistance due to the secondary T790Mmutation in NSCLC by inducing enhanced cytotoxicityboth in vitro and in vivo [21]. Furthermore, the associationof cetuximab with afatinib has been shown to be effectiveto overcome T790M-mediated drug resistance [22].

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However, the combination of erlotinib with cetuxi-mab did not lead to a significant radiological responsein NSCLC patients with clinically defined acquiredresistance to erlotinib indicating that such strategy isnot sufficient to overcome acquired resistance to erlo-tinib [23].The mechanisms leading to an enhanced activity of

combining a TKI with a monoclonal antibody havebeen ascribed, in other cancer cell models, either to amore efficient inhibition of TK receptors [17] or toan increased targeted receptors on plasma membraneinduced by TKIs [24,25]. Scaltriti et al. showed thatlapatinib enhanced the effects of trastuzumab by in-ducing HER-2 stabilization and accumulation at thecell surface of breast cancer cell lines [24], andMimura et al. reported that lapatinib induced accumu-lation of HER-2 and EGFR on esophageal cancer celllines evoking trastuzumab- and cetuximab- mediatedADCC [25].ADCC, one of the killing mechanism of the immune

system mediated by Natural Killer cells, plays a pivotalrole in the anti-cancer effects exerted by mAbs. There-fore, increasing the ADCC activity is an importantobjective in the development of novel therapeuticapproaches.It has been recently demonstrated that the EGFR inhi-

bitors gefitinib and erlotinib enhance the susceptibilityto NK cell mediated lysis of A549, NCI-H23 and SW-900 lung cancer cell lines [26] by the induction ofULBP1 (a ligand of the NK cell activation receptorNKG2D). These data indicate that EGFR blockade couldnot be the only mechanism of action of EGFR inhibitorsin vivo. The efficacy of these inhibitors in lung cancermay be at least in part mediated by increased suscepti-bility to NK activity. Moreover, cetuximab serves as apotent stimulus for NK functions including INF-gammaproduction [27] and is also associated with a comple-ment –mediated immune response [28].We here demonstrated that erlotinib induces an accu-

mulation of EGFR and/or HER2 protein at the plasmamembrane level only in TKI sensitive NSCLC cell lineswhereas, in resistant cells (both, intrinsic or METamplification-mediated acquired resistance), this en-hancement was not observed. The anti-tumour effect ofdrug combination was more evident in ADCC experi-ments compared with cell viability experiments. In theCalu-3 xenograft model, the combined treatmentresulted in a lower rate of tumour growth, suggestingthe involvement of NK activity as a determinant factorto improve the efficacy of the combined treatment.Moreover, regressive phenomena and changes in size ofneoplastic glands together with intense stromal reactionwere observed in histologic samples of tumours frommice treated with cetuximab alone or the combination.

The reason why EGFR inhibitors such as gefitinib,erlotinib or lapatinib induce EGFR accumulation only insensitive cells could be ascribed to their ability to inhibitsignal transduction pathways downstream EGFR. Theconstitutive activation of signaling pathways downstreamof EGFR (i.e. presence of RAS mutations) is indeed arecognized mechanism of resistance against reversibleEGFR-TKIs [29]. The inhibition of the MAPK pathwaymight represent a link between EGFR inhibition andEGFR accumulation since U0126, a well known MEK1/2inhibitor, induced EGFR accumulation in Calu-3 cells,while none of PI3K/AKT/mTOR inhibitors tested waseffective. A correlation between MAPK pathway andprotein degradation by the ubiquitin system wasdescribed for the pro-apoptotic BH3-only protein BIM,indeed in the absence of MAPK activation, BIM proteinaccumulated in the cell promoting activation of apop-totic cell death [30].Considering that EGFR TKIs, in particular erlotinib,

demonstrated to be effective only in a small percentageof NSCLC patients not harboring EGFR mutations,our preclinical results could support clinical trials onthe combinations of erlotinib and cetuximab or trastu-zumab aiming to improve treatment efficacy. Althoughthe addition of cetuximab to erlotinib is insufficient toovercome erlotinib resistance in EGFR-driven lungadenocarcinoma [23], the clinical potential of dual-agent molecular targeting of the EGFR in patients withEGFR wild-type tumours remains to be elucidated andmay represents an interesting research area to bepursued.

ConclusionsIn this study we explored the potential of combiningerlotinib with cetuximab or trastuzumab in improvingthe efficacy of EGFR targeted therapy in EGFR wild-typeerlotinib-sensitive NSCLC cell lines. Our results indicatethat erlotinib, through ERK inhibition, increases surfaceexpression of EGFR and/or HER2 only in erlotinib sensi-tive NSCLC cell lines and in turn leads to increased sus-ceptibility to ADCC both in vitro and in xenograftsmodels.These data prompt future adequate clinical trials that

will give the ultimate proof of the utility of this com-bined treatment for the care of NSCLC patients carryingEGFR-wild type that are sensitive to TKIs.

MethodsCell cultureThe human NSCLC cell lines H322, H292, Calu-3, H1299,A549, H1703 and Calu-1 were obtained from AmericanType Culture Collection (Manassas, VA, USA) and werecultured as recommended. The PC9, HCC827 andHCC827GR5 cell lines were kindly provided by Dr P.

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Jänne (Dana-Farber Cancer Institute, Boston MA, USA).All cells were maintained under standard cell cultureconditions at 37°C in a water-saturated atmosphere of 5%CO2 in air. As previously reported [31] cells showing byproliferation assays IC50 for erlotinib < 1 μM were consid-ered sensitive (H322, H292, Calu-3, PC9, HCC827) whilecell lines with IC50 > 5 μM (H1299, A549, H1703, Calu-1,HCC827GR5) were considered resistant.

Drug treatmentErlotinib, gefitinib, cetuximab, trastuzumab and rituximabwere from inpatient pharmacy. RAD001, NVP-BKM-120and NVP-BYL-719 were from Novartis.Stock solutions of 20 mM drugs were prepared in

dimethylsulfoxide (DMSO) (with the exception ofmAbs), stored at −20°C and diluted in fresh medium foruse. The final concentration of DMSO never exceeded0.1% v/v.

Western blot analysisProcedures for protein extraction, solubilization, andprotein analysis by 1-D PAGE are described elsewhere[32,33]. Fifty μg of proteins from lysates were resolvedby 7.5% SDS-PAGE and transferred to PVDF mem-branes. Membranes were incubated with: 1:1000 rabbitpolyclonal anti-EGFR; 1:1000 rabbit mAb anti-HER2/ErbB2; 1:1000 rabbit mAb anti-Phospho-p70S6K(Thr421/Ser424); 1:1000 mouse mAb anti-Phospho-p44/42 MAPK (ERK1/2); 1:1000 rabbit mAb anti-p44/42MAPK (ERK1/2) (Cell Signaling Technology, Beverly,MA, USA); 1:1000 mouse mAb anti- Transferrin Receptor(Invitrogen Corporation, Camarillo, CA, USA); 1:3000mouse mAb anti-Actin (Sigma–Aldrich, St Louis, MO,USA). Blots were then washed and incubated with HRP-anti-mouse or HRP-anti-rabbit antibodies at 1:20000 dilu-tion (Pierce, Rockford, IL, USA). Immunoreactive bandswere visualized using an enhanced chemiluminescencesystem (ImmobilionTM Western Cemiluminescent HRPSubstrate, Millipore USA).

Cell surface protein isolationCalu-3 cells were grown in T75 flasks and treated with0.5 μM erlotinib for 24 h. Cells were incubated withEZ-LINK Sulfo-Biotin (Pierce) for 2 h at 4°C with gentlerotation. The reaction was stopped by washing twicewith 25 mM Tris–HCl (pH 7.5) in PBS (phosphate-buf-fered saline) and cells were scraped into ice-cold lysisbuffer (50 mmol/l HEPES, pH 7.0, 10% glycerol, 1% Tri-tonX-100, 5 mmol/l EDTA (ethylenediaminetetraaceticacid), 1 mmol/l MgCl2, 25 mmol/l NaF, 50 μg/ml leu-peptin, 50 μg/ml aprotinin, 0.5 mmol/l orthovanadate,and 1 mmol/l phenylmethylsulfonyl fluoride). Lysateswere centrifuged at 15000 g for 20 min at 4°C, andsupernatants were removed and assayed for protein

concentration using the DC Protein assay (Bio-Rad, CA,USA). A volume of 500 μl of lysis buffer containingequal amount of proteins was incubated with UltraLinkImmobilized NeutrAvidin protein (Pierce) for 2 h at 4°Cwith gentle rotation, washed three times with lysis bufferbefore suspension in SDS (sodium dodecyl sulfate)-load-ing buffer and then resolved by SDS-PAGE.

Flow cytometryFor the determination of EGFR and HER2 protein mem-brane levels, NSCLC cell lines H322, Calu-3 and H292were treated with 1 μM erlotinib for 24 h. One millioncells per condition were then incubated with Isotypecontrol Monoclonal Mouse IgG1/R-PE (Ancell IRP,Bayport, MN, USA), PE mouse anti-Human EGFR(Calu-3 and H322 cells) (BD Biosciences, San Josè, CA,USA) or PE mouse anti-Human HER2 (H322 and H292)(BD Biosciences). After the incubation the analysis wasperformed with an EPICS-XL flow cytometer.For the relative quantization of EGFR or HER2 bind-

ing sites, NSCLC cell lines H322, Calu-3, H292 weretreated with 1 μM erlotinib for 24 h. One million cellswere then dispensed for each condition and treated witheither 20 μg/ml rituximab (Isotype control), cetuxi-mab (Calu-3 and H322) or trastuzumab (H292) for1 h. After the incubation with PE-anti-human-IgG(BD Biosciences), the analysis was performed with anEPICS-XL flow cytometer.The values of mean fluorescence intensity (MFI) were

converted in units of equivalent fluorochrome (MEF)using the FluoroSpheres 6-Peak Kit (Dako, CA, USA).

Quantitative real-time PCRTotal RNA was isolated by the TRIzolW reagent (Invitrogen,Carlsbad, CA, USA) and reverse transcribed as previouslydescribed [33].The transcript levels of EGFR gene were assessed

by Real-Time qRT-PCR on an iCycler iQ MulticolorRealTime PCR Detection System (Bio-Rad, Hercules,CA, USA).Primers and probes included: EGFR-F (50-GCCTT

GACTGAGGACAGCA-30), EGFR-R (5-TTTGGGAACGGACTGGTTTA-3), EGFR-probe (50-FAM CTTCCTCC30DQ); PGK1-F (50-GGAGAACCTCCGCTTTCAT-30), PGK1-R (50-CTGGCTCGGCTTTAACCTT-30), PGK1-probe (50-FAM GGAGGAAG 30DQ); RPL13-F (50-ACAGCTGCTCAGCTTCACCT-30), RPL13-R (50-TGGCAGCATGCCATAAATAG-30), RPL13-probe (50-FAMCAGTGGCA30DQ); HPRT-F (50-TGACCTTGATTTATTTTGCATACC-30), HPRT-R (50CGAGCAAGACGTTCAGTCCT-30), HPRT-probe (50-FAM GCTGAGGA 30DQ).The amplification protocol consisted of 15 min at

95°C followed by 40 cycles at 94°C for 20s and at 60°C for1 min.

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The relative transcript quantification was calculatedusing the geNorm algorithm for Microsoft ExcelTM

after normalization by expression of the control genes[phosphoglycerate kinase1 (PGK1), ribosomal proteinL13 (RPL13) and hypoxanthine-guanine-phosphoribo-syltransferase (HPRT)] and expressed in arbitraryunits (a.u.).

MTT assayThe cells were seeded into 96-well plate in quadruplicateand were exposed to various treatments. After 96 h,100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetra-zolium bromide (MTT) solution (1 mg/ml, Sigma-Aldrich) was added to each well and incubated. After4 h, crystalline formation was dissolved with DMSO andthe absorbance at 570 nm was measured using themicroplate-reader 550 (BioRad).

Isolation and culture of NK cellsHuman PBMC were isolated from buffy coat ofhealthy donors by using a Lympholyte-H density gra-dient centrifugation (Cederlane Burlington, Ontario,Canada). Highly purified CD56+ natural killer (NK)cells were obtained by magnetic separation using theNK Cell Isolation Kit and the autoMACS Separator(Miltenyi Biotec, Cologne, Germany) according to theuser manual.Purified NK cells were resuspended in culture medium

(RPMI 1640 without phenol red and supplemented withheat inactivated 10% FCS, 50 U/ml penicillin, 50 U/mlstreptomycin, 2 mmol/l glutamine) plated and preincu-bated at 37°C for up to 18 h in the presence of humanInterleukin-2 (IL-2, 100 U/ml, Miltenyi Biotec).

ADCC assayAntibody-dependent cell-mediated cytotoxicity (ADCC)was measured with the CytoTox 96 non-radioactivecytotoxicity assay (Promega, Madison, WI, USA) accord-ing to manufacturer’s instructions. 2x103 Calu-3, H322,H292 or H1299 cells were treated for 24 h with 1 μMerlotinib, and then seeded with purified NK cells (ratioof 1:25 and 1:50) in a 96-well plate and incubated with10 μg/ml cetuximab or trastuzumab. After 4 hours thelactate dehydrogenase (LDH) release was determinedand the percentage of cytotoxicity was calculated aftercorrecting for background absorbance values accordingto the following formula:

%Cytotoxicity¼Experimental�Effector spontaneous�Target maximum�Target s

Tumour xenograftsAll experiments involving animals and their care wereperformed with the approval of the Local EthicalCommittee of University of Parma, in accordance withthe institutional guidelines that are in compliance withnational (DL116/92) and international (86/609/CEE)laws and policies. Twenty-four Balb/c-Nude female mice(Charles River Laboratories, Calco, Italy) were housed ina protected unit for immunodeficient animals with12-hour light/dark cycles and provided with sterilizedfood and water ad libitum. At the time of xenograft es-tablishment, mice were 8 weeks old and weighted ~20g.200 μl of matrigel (BD Biosciences) and sterile PBS (1:1)containing 1x107 Calu-3 cells, were subcutaneouslyinjected on the right flank of each mouse (using a 1 mlsyringe, needle G25). When tumour volume reached anaverage size of 300 mm3, 14 days after injection, animalswere randomized into four groups and the treatmentstarted. After 4 weeks, mice were euthanized by cervicaldislocation and tumours collected for immunohisto-chemistry and histological analysis.Erlotinib (25 mg/Kg) was administered orally in 1%

methylcellulose, 0.2% Tween 80 in sterilized water5 days/week. Cetuximab (2 mg/Kg) was intraperitoneallyinjected in sterile saline solution 2 days/week. Controlgroup received both oral gavage of 1% methylcellulose,0.2% Tween 80 in sterilized water 5 days/week and i.p.injection of sterile saline solution (0.9%) 2 days/week.Dosages of drugs were chosen halving the one used in

a previous study in NSCLC-xenograft models, in orderto avoid the complete inhibition of tumour growth bythe single agent treatment and to better highlight theeffect of erlotinib-cetuximab combination [19,34].Tumour xenografts were measured twice a week,

tumour volume was determined using the formula:(length x width2)/2. Final data are expressed as percentof volume increase: (tumour volume/pre-treatmenttumour volume) x 100.

Morphometric and immunohistochemical analysis oftumour xenograftsFormalin fixed samples were embedded in paraffin. Fromeach tumour serial sections of 5 μm thickness wereobtained and stained with Haematoxylin and Eosin (H&E),Masson’s Trichrome and for immunohistochemistry.Morphometric analysis was performed in order to evaluate:(a) the numerical density of neoplastic cells, (b) the volumefraction of interstitial inflammatory cells, (c) the volume

Target spontaneouspontaneous

�100

Cavazzoni et al. Molecular Cancer 2012, 11:91 Page 13 of 14http://www.molecular-cancer.com/content/11/1/91

fraction of fibrosis and (d) the fraction of proliferating andapoptotic cells.In particular, for each section stained with H&E, a

quantitative evaluation of tissue composition was per-formed. To better define the fraction occupied byneoplastic and non neoplastic cells, sections werestained with pancytokeratin antibodies (monoclonalmouse, 1:500, o.n. 4°C, Dako) revealed through biotin-streptavidin-DAB system, as repeatedly described. Thenumerical density (n/mm2) of pancytokeratin positiveneoplastic cells was computed.In addition, cell proliferation and apoptotic death were

investigated by fluorescence microscopy. Thus, Ki67 label-ing (rabbit polyclonal antibody, Vector) and the Terminaldeoxynucleotidyltransferase (TdT)–mediated dUTP nickend labeling (TUNEL) assay (Roche Diagnostics, Italy) oncytokeratinpos neoplastic cells were revealed by specificfuorescent probes.The area occupied by interstitial cells was expressed as

percentage of the total area explored. By the same ap-proach, the volume fraction of fibrosis was calculated onMasson’s Trichrome stained sections. To define thevolume fractions, the number of points overlying eachtissue components was counted and expressed as per-centage of the total number of points explored.All these morphometric measurements were obtained

with the aid of a grid defining a tissue area of 0.23 mm2

and containing 42 sampling points each covering an areaof 0.0052 mm2.All these evaluations were performed on the entire

section of each tumour sample of each experimentalgroup of animals using an optical microscope (250Xfinal magnification).

Statistical analysisStatistical analyses were carried out using GraphPadPrism version 5.0 software (GraphPad Software Inc.,San Diego, CA, USA). Results are expressed as meanvalues ± standard deviations (SD) for the indicatednumber of independent measurements. Differences be-tween the mean values recorded for different experi-mental conditions were evaluated by Student’s t-test,and P values are indicated where appropriate in the fig-ures and in their legends. A P value <0.05 was consid-ered as significant.For in vivo studies comparison among groups was

made using analysis of variance (two-way ANOVArepeated measures) followed by Bonferroni’s post-test.Analysis was performed using Prism 5.0 (GraphPad Soft-ware) and differences were considered significant whenP value was below 0.05. The nature of the interactionbetween erlotinib and cetuximab was calculated usingthe Bliss interaction model [35].

AbbreviationsADCC: Antibody-dependent cellular-cytotoxicity; EGFR: Epidermal growthfactor receptor; MAPK: Mitogen-activated protein kinase; NSCLC: Non smallcell lung cancer; TKI: Tyrosine kinase inhibitor.

Competing interestAll authors declare that they have no competing interests.

Authors’ contribution

AC carried out ADCC experiments, interpreted the results and assisted with thedraft of the manuscript; DC isolated and cultured NK cells and carried out flowcytometry analysis; FS, LA and PC performed the in vivo studies; PM carried outRT-PCR experiments; MG and MB carried out Western blot analysis; EGperformed the statistical analysis, SLM and CF carried out cell growthexperiments; FQ, GG and DM carried out immunohistochemical analysis; AM,MT, EB and PGP critically revised the manuscript; AA designed the project andassisted with the draft of the manuscript; RRA, analyzed the results and wrotethe manuscript. All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by: Associazione Italiana per la Ricerca sul Cancro(AIRC), Milan grant IG 8856; Associazione Augusto per la Vita (Novellara, RE);Associazione Davide Rodella, Montichiari, BS; Associazione Chiara Tassoni,Parma; A.VO.PRO.RI.T., Parma.

Author details1Department of Clinical and Experimental Medicine, University of Parma,Parma, Italy. 2Department of Surgical Science, University of Parma, Parma,Italy. 3Italian Workers’ Compensation Authority (INAIL) Research Center,University of Parma, Parma, Italy. 4Division of Medical Oncology, UniversityHospital of Parma, Parma, Italy. 5Department of Pharmacy, University ofParma, Parma, Italy.

Received: 30 August 2012 Accepted: 3 December 2012Published: 12 December 2012

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doi:10.1186/1476-4598-11-91Cite this article as: Cavazzoni et al.: Combined use of anti-ErbBmonoclonal antibodies and erlotinib enhances antibody-dependentcellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines.Molecular Cancer 2012 11:91.

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